αSMA-Expressing Perivascular Cells Represent Dental Pulp Progenitors In Vivo.

The goal of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin using mouse molars as a model. We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. In vivo lineage-tracing experiments in molars showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. This population also makes a small contribution to odontoblasts during primary dentinogenesis.

Gene-expression analysis of cementoblasts and osteoblasts.

BACKGROUND AND OBJECTIVE: Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. MATERIAL AND METHODS: OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. RESULTS: Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. CONCLUSION: The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Carotid artery intima thickness and flow velocity after discontinuation of hormone replacement therapy in postmenopausal women: follow-up study.

AIM: To investigate the effect of discontinuation of hormone replacement therapy on the intima thickness and blood flow velocity of the common carotid artery. METHODS: The thickness of the left common carotid artery intima and maximal systolic blood velocity were measured by real-time and Doppler ultrasonography in 75 healthy postmenopausal women starting sequential combined hormone replacement therapy. The measurements were performed at the start and after 6 and 12 months of the therapy. Thirty two women decided to discontinue the therapy after 6 months, whereas 43 continued. RESULTS: In the group that continued with the hormone replacement therapy, a significant decrease was recorded in the mean baseline values for carotid artery intima thickness and flow velocity at 6 months (0.35+0.11 vs 0.54+0.19 mm and 0.73+0.16 vs 0.87+0.19 m/s, respectively, p?0.001) and 12 months of follow-up (0.36+0.1 mm and 0.72+0.15 m/s vs baseline, respectively, p?0.001). In women who discontinued the therapy, there were significant deviations from the baseline values in the intima thickness (0.36+0.11 vs. 0.59+0.09 mm, p=0.010) and flow velocity (0.75+0.14 vs. 0.85+0.16 m/s, p?0.001) at 6 but not at 12 months of the follow-up (0.55+0.12 mm, and 0.85+0.17 m/s vs baseline; p=0.148 and p=0.965, respectively). CONCLUSION: Decreased flow velocity and reduced intima thickness were directly related to blood vessel wall dilatation after estrogen component of hormone replacement therapy. Discontinuation of the hormone replacement therapy returned the flow velocity and intima thickness to their baseline values.

Evolution of clinical symptoms in a young woman with a recurrent gonadotroph adenoma causing ovarian hyperstimulation.

OBJECTIVE: To demonstrate the clinical course in a young female with gonadotroph adenoma causing ovarian stimulation. PATIENT AND METHODS: Our patient was a 23-year-old woman with a history of oligomenorrhea who had previously undergone bilateral ovarian wedge resection owing to the clinical appearance of polycystic ovaries. Two years later, she sought treatment for headache, galactorrhea, history of spotting and lower abdominal distension. FSH, LH, beta-LH, inhibin A and B, estradiol, prolactin (PRL), and beta-chorionic gonadotrophin (beta-CG) were measured, and the responses of FSH, LH and beta-LH to thyrotrophin-releasing hormone (TRH) were documented. Immunohistochemical analysis of the tumor tissue was performed after surgery. Five years after the trans-sphenoidal surgery, the patient again became oligomenorrheic. A large recurrent adenoma was diagnosed on CT one year later. Transvaginal ultrasound showed ovaries of normal size with multiple small cystic formations simulating a polycystic pattern, While the patient was awaiting surgery, a pituitary apoplexy occurred. Emergency decompressive surgery was performed and the patient fully recovered. RESULTS: Enlarged ovaries were found on ultrasound examination simulating a hyperstimulation-like pattern. At that time, elevated levels of FSH (13.4IU/l) and marginally elevated levels of beta-LH (1.43ng/ml) were found, whereas the level of LH (0.5IU/l) was subnormal. Plasma estradiol was markedly supranormal (6150pmol/l). Levels of inhibin A and B were elevated (326pg/ml and 588pg/ml respectively). The prolactin level (70ng/ml) was increased, whereas beta-chorionic gonadotrophin (beta-CG) was normal. Significantly increased FSH, LH, and beta-LH responses to TRH stimulation were documented. Pituitary macroadenoma was found on MRI scan and removed by trans-sphenoidal surgery. Immunohistochemical examination showed high positivity for beta-CG and LH, and slight positivity for FSH. Five years after the surgery, estradiol was elevated (1160pmol/l), whereas basal levels of LH (4.65IU/l) and FSH (3.98IU/l) were not suppressed. After the second operation, immunostaining of the adenoma tissue confirmed the previous findings. CONCLUSIONS: Measurement of gonadotrophins in our case did not prove to be a method for identifying a large recurrent gonadotroph pituitary adenoma. The sonographic ovarian imaging varied from a polycystic- to an ovarian hyperstimulation-like pattern during the evolution of the tumour.

Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment.

We have determined the crystal structure of a spliceosomal RNP complex comprising the 15.5kD protein of the human U4/U6.U5 tri-snRNP and the 5' stem-loop of U4 snRNA. The protein interacts almost exclusively with a purine-rich (5+2) internal loop within the 5' stem-loop, giving an unusual RNA fold characterized by two tandem sheared G-A base pairs, a high degree of purine stacking, and the accommodation of a single RNA base, rotated out of the RNA chain, in a pocket of the protein. Apart from yielding the structure of an important entity in the pre-mRNA splicing apparatus, this work also implies a model for the complex of the 15.5kD protein with box C/D snoRNAs. It additionally suggests a general recognition principle in a novel family of RNA binding proteins.

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.

[The level of reliability of Lasegue's symptoms measured in the sitting position--results of a multicenter study]. / Stupanj pouzdanosti Lasegue-ovog simptoma mjerenog u sjedecem polozaju--rezultati multicentrickog ispitivanja.

The results of Lasègue test /LT/ examination are not always enough objective owing to subjective complaints superposition. The first favourable clinical experiences with analysis of the LT in sitting position stimulated our previous anatomical, biometrical and clinical investigations (Mandic and col. 1970.). There has been established the stretching of the ischial nerve in classical LT to 12.7 cm, in sitting position to 4.8 cm, and the changing of position from supine to sitting to 6.4 cm. That means that the stretching in sitting LT is greater then in classical way/i.e. 13.26 cm/. The stretching forces in both ways were equal (0-3.7 kp/. The multicentric investigation included 330 patients of both sexes, aged 15-62 years. In 57 patients/17.3%/was LT in sitting position positive and in supine negative. That demonstrate the higher degree of reliability 17.3%. These quantitative results will be continued in qualitative investigation of statistical correlation of relative flexion degrees. The advantages of sitting LT are:more simple, more quick, the taking off patient's clothes is not needed, and the result is more suitable for the objective evaluation of psychogenic complaints superposition or simulation.